This invention is directed to a process for the preparation of peptides and peptide amides which contain sulfated tyrosine or sulfated hydroxy amino acids using benzotriazol-1-yl-oxy-tris (dimethyl)-phosphonium hexafluoro-phosphate (BOP) as a coupling reagent. In the method of the invention the need to use amino acid protecting groups to preclude unwanted side reactions during peptide synthesis is minimized. The instant invention is particularly directed to the solid phase synthesis of potent CCK-8 analogs using BOP as a coupling reagent.
Cholecystokinin (CCK) is a polypeptide hormone which was first isolated as a 33-amino acid peptide from the porcine gastrointestinal tract. [Mutt et al., Biochem. J. 125. 57-58 (1971) and Mutt et al., Clin. Endocrinol. supplement, 5, 175-183 (1976)]. Peripherally administered CCK has been shown to produce satiety in the rat and the monkey and infusions of CCK-8, the octapeptide analog of CCK, has been shown to decrease food intake in lean and obese men. G.P. Smith, Int. J. Obesity 8 Suppl. 1:35-38 (1984); Jorpes et al., Acta. Chem. Scand. 18:2408 (1964); Della-Fera et al., Science 206:471-73 (1979); Gibbs et al., J. Comp. Physiology and Psychology 84, 488-495 (1973). It is now accepted that CCK has satiety-inducing effects and thus, may be useful to reduce or suppress food intake in man.
The polypeptide hormone, CCK-33, has the amino acid sequence:
Lys-Ala-Pro-Ser-Gly-Arg-Val-Ser-Met-Ile-Lys-Asn-Leu-Gln-Ser-Leu-Asp-Pro-Ser -HisArg-Ile-Ser-Asp-Arg-Asp-Tyr-(SO.sub.3 H)-Met-Gly-TrpMet-Asp -Phe-NH.sub.2. PA1 (a) preparation of the .alpha.-amino acid protected O-CH.sub.2 -PAM-resin according to known methods. PA1 (b) sequential addition of side-chain unprotected t-butyloxycarbonyl (Boc) hydroxyamino acid using the BOP reagent. In general, single couplings and 3 equivalents of BOP and three equivalents of Boc amino acids are used. PA1 (d) cleavage of the multi-sulfated peptide with a nucleophilic agent selected from the group consisting of ammonia, amines or hydrazine. PA1 (e) single step purification of the multi-sulfated peptide by semi-preparative HPLC.
Fragments of CCK, e.g. CCK-8 and CCK-7 also have been shown to have satiety-inducing effects. CCK-8 has the amino acid sequence: EQU Asp-Tyr-(SO.sub.3 H)-Met-Gly-Trp-Met-Asp-Phe-NH.sub.2
CCK-7 is one amino acid less than CCK-8, i.e., it is CCK-8 minus the 26-position Asp.
Ondetti and Pluscec [J. Am. Chem. Soc. 92. 195 (1970); J. Med Chem. 13, 349 (1970); see also U.S. Patent Nos. 3,723,406; 3,778,429; 3,835,315 and 3,892,726], synthesized CCK-8 and a series of analogs by the method of solution phase peptide synthesis. Toth and Kovacs [Int. J. Peptide Protein Res. 26, 630 (1985)]evaluated four different strategies for preparing CCK-8 peptides using solution phase synthesis. Penke et al. (U.S. Pat. No. 4,102,878) described a process whereby CCK-8 was produced by the known method of solution phase peptide chemistry through an improved sulfation procedure using pyridinium acetyl sulfate (PAS) as the sulfation agent. Penke and Rivier [J. Med Chem. 27. 845 (1984)]described the synthesis of unsulfated analogs of CCK-8 using solid phase peptide methodology by the standard procedure generally described by Merrifield [J. Am. Chem. Soc. 85, 2149 (1963)]. The non-sulfated heptapeptide analogs were synthesized on methylbenzhydrylamine-resin with t-butyloxyCarbonyl (Boc) protected amino acids. The beta carboxy group (.beta. -COOH) of the amino acid of Asp was protected with 0-Benzyl, and the hydroxy group of Ser, Thr, Hyp was protected with the benzyl group. The phenolic group of the amino acid Tyr was protected with the 2,6-dichlorobenzyl group. Other amino acid side chains were unprotected. Each coupling was made with dicyclohexyl-carbodimide (DCC). N-terminal acetylation was performed on the resin using acetic anhydride. The peptides were cleaved from the resin with hydrogen fluoride in the presence of scavengers, purified by high performance liquid chromatography (HPLC) and sulfated with PAS.
Comstock and Rosamond in European Patent 161468 described the solid phase synthesis of peptides containing sulphated tyrosine by (a) preparing a peptidyl derivative of an aminomethyl polystyrene resin by attachment of a 4-(ROCH.sub.2)Phenylacyl group (R=acyl derived from protected amino acid) followed by sequential addition of active esters of protected amino acids; (b) converting tyrosine groups in the peptidyl resin to their sulfate esters by reaction with pyridine sulfur trioxide complex or (PAS); (c) cleaving the peptide from the resin by treatment with alkali, NH.sub.3, aliphatic amines, or hydrazine. Penke and Rivier [J. Org. Chem. 52, 1197-1200 (1987)]described a new route for the synthesis of CCK-8 and analogs utilizing 2,4-dimethoxybenzhydrylamine support in conjunction with the fluoroylmethyloxy carbonyl tert-Butyl (Fmoc/tBu) strategy with trifluoroacetic acid as a cleavage reagent for removal of the peptide from the resin.
All the above mentioned processes utilize DCC, DCC/HOBt or symmetrical anhydride as the coupling reagents, however these reagents are not without shortcomings [Bodanszky (1984), Principles of Peptide Synthesis, Springer Verlag, New York], and side-reactions such as racemization or intramolecular rearrangement. Repetitive couplings are often required for the complete introduction of a residue in the peptide chain during the synthesis. Among several coupling reagents suggested for replacement of DCC, the "BOP reagent" [benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluoro-phosphate] proposed by Castro, Tet. Lett., 14 1219-1222, (1975) represents an attractive alternative. This is a non-hygroscopic salt, very stable and soluble in the usual organic solvents used in peptide synthesis. The activation of the carboxy (COOH) function is reported to proceed via the formation of an acyloxyphosphonium derivative, subsequently transformed into the HOBt active ester, which undergoes rapid coupling with the growing peptide chain.
The BOP reagent has been used in solution synthesis [Castro, J. Chem. Soc. Perkin Trans. I, 1025-1031 (1985)] and has been applied/in solid phase for fragment coupling [Rivaille, Tet. 36, 3413-3419 (1980)]and stepwise synthesis [Martinez, FEBS Lett. 200, 181-185 (1986)]. However, the BOP reagent has always been used in solid phase synthesis in conjunction with fully protected trifunctional amino acids. The process described in this invention utilizes the BOP reagent for the solid phase coupling of unprotected aliphatic and aromatic hydroxyamino acids.